Method for Simultaneously Separating and Purifying Two Galloylated Myricitrins from Morella rubra Leaves, and Use therefor

Abstract

A method for simultaneously separating and purifying myricetin-3-O-(2″-O-galloyl)-a-L-rhamnoside and myricetin-3-O-(4″-O-galloyl)-a-L-rhamnoside from Bayberry leaves, and the use of the compounds prepared by the method. The method includes: (1) alcohol extraction and concentration: mixing Bayberry leaves with an alcohol solution; (2) solid-phase extraction column adsorption: injecting the crude Bayberry leaf flavonol alcohol extract into a solid-phase extraction column; washing with a mobile phase for the first gradient elution; (3) preparative liquid chromatography purification: using a solid-phase chromatography column; wherein the solid-phase extraction powder obtained from step (2) is subjected to a second gradient elution with a mobile phase and then processed to obtain the target products: myricetin-3-O-(2″-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside monomer.

What is claimed is: 
     
         1 . A method for simultaneously separating and purifying myricetin-3-O-(2″-galloyl)-α-L-rhamnoside and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside from Bayberry leaves, comprising the following steps:
 (1) alcohol extraction and concentration: mixing Bayberry leaves with an alcohol solution; extracting by ultrasound; filtering and collecting the filtrate; removing the alcohol from the filtrate and concentrating to obtain a crude Bayberry leaf flavonol alcohol extract; wherein the volume percentage of the alcohol solution is 50-100%; 
 (2) solid-phase extraction column adsorption: injecting the crude Bayberry leaf flavonol alcohol extract into a solid-phase extraction column; washing with a mobile phase for the first gradient elution; and processing the collected eluent to obtain a solid-phase extraction powder rich in myricetin-3-O-(2″-galloyl)-α-L-rhamnoside and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside; 
 the first gradient elution process is as follows: first eluting with an alcohol solution with a volume percentage lower than 40% for the first elution; then using an alcohol solution with a volume percentage of 40-60% for the second elution; and collecting the eluent after the second elution; wherein the concentration of the alcohol solution for the first elution is above 20% by volume percentage; 
 (3) preparative liquid chromatography purification: using a solid-phase chromatography column; wherein the solid-phase extraction powder obtained from step (2) is subjected to a second gradient elution with a mobile phase and then processed to obtain the target products: myricetin-3-O-(2″-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside monomer; 
 the mobile phase: Phase A is selected from a formic acid-water solution with a volume percentage of 0.05-5%, a trifluoroacetic acid-water solution with a volume percentage of 0.05-5%; Phase B is selected from an acetonitrile-water solution with a volume percentage of 40-60%, an acid-acetonitrile-water solution with a volume percentage of 40-60%; the volume percentage of the acid is 0.05-5%, and the acid is selected from formic acid, trifluoroacetic acid; 
 the second gradient elution process is as follows: the volume percentage of Phase B increases from 20% to 60% within 0-10 minutes, from 60% to 90% within 10-30 minutes, and from 90% to 100% within 30-35 minutes; and then decreases from 100% to 20% within 35-40 minutes, collecting the target products separately; 
 the alcohol solution refers to an alcohol aqueous solution, and the alcohol is selected from methanol or ethanol. 
 
     
     
         2 . The method according to  claim 1 , wherein in step (1), the filtrate is concentrated by vacuum rotary evaporation at 37-50° C. to remove the alcohol and obtain the crude Bayberry leaf flavonol alcohol extract. 
     
     
         3 . The method according to  claim 1 , wherein in step (1), the process of collecting the filtrate is repeated 2-4 times, and the filtrates of 2-4 times are combined. 
     
     
         4 . The method according to the method of  claim 1 , wherein in step (2), the gradient elution of the solid phase extraction column is as follows:
 the crude Bayberry leaf flavonol alcohol extract is injected into the solid-phase extraction column, washed with deionized water; and gradient elution is performed with a mobile phase; and the collected eluent is vacuum rotary evaporated at 37-45° C. to obtain the solid phase extraction powder rich in the target product.   
     
     
         5 . The method according to the method of  claim 1 , wherein in step (2), the adsorption of the solid phase extraction column is as follows:
 the C18 Sep-Pak® solid phase extraction column is activated, and 4.5 BV of Bayberry leaf extract is applied to each column; 4 BV of deionized water is used to wash away saccharic acid; then 10 BV of a 30% methanol solution is used to elute, removing some impurities and other non-target flavonols; then 4 BV of a 40% methanol solution is used to elute, collecting the eluent of the 40% fraction, and vacuum rotary evaporated at 37-50° C. to obtain the solid phase extraction powder rich in myricetin-3-O-(2″-galloyl)-α-L-rhamnoside and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside.   
     
     
         6 . The method according to  claim 1 , wherein in step (3), Phase B is selected from a 50% acetonitrile-water solution, a 50% acid-acetonitrile-water solution; the volume percentage of the acid is 0.1-5%; and the acid is selected from formic acid, trifluoroacetic acid. 
     
     
         7 . The method according to  claim 1 , wherein in step (3), the preparative liquid chromatography purification is as follows:
 a preparative liquid column SunFire™ C18 OBM™ column (5 μm, 19×250 mm) is used; the mobile phase: Phase A: a 0.1% formic acid-water solution, Phase B: a 50% formic acid-acetonitrile-water solution (wherein, the volume percentage of formic acid is 0.1%); the column temperature is room temperature, and the flow rate is 3-6 mL/min;   the solid phase extraction powder obtained in step (2) is dissolved in methanol to a concentration of 100-200 mg/mL, injected into the preparative liquid phase for purification, with a single injection volume of 50-300 μL;   the eluate is collected separately at 27-28.5 minutes and 28.5-30 minutes, and the eluate rich in pure target product is combined, and then concentrated and freeze-dried to obtain high purity myricetin-3-O-(2″-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside monomer.   
     
     
         8 . The method according to  claim 1 , wherein 30%-40% alcohol solution is used for the first elution. 
     
     
         9 . The method according to  claim 1 , wherein Phase A is selected from a formic acid-water solution with a volume percentage of 0.1-3%. 
     
     
         10 . The method according to  claim 1 , wherein in step (3), the preparative liquid column SunFire™ C18 OBM™ column (5 μm, 19×250 mm) is used; and during the second gradient elution, the eluate is collected separately at 27-28.5 minutes and 28.5-30 minutes; the column temperature is room temperature, and the flow rate is 3-6 mL/min. 
     
     
         11 . The method according to  claim 1 , wherein the solid phase extraction powder obtained in step (2) is dissolved in methanol to a concentration of 100-200 mg/mL, injected into the preparative liquid phase for purification, with a single injection volume of 50-300 μL. 
     
     
         12 . The method according to  claim 1 , wherein the volume percentage of the alcohol solution is 80%. 
     
     
         13 . The method according to  claim 1 , wherein the ultrasound extraction time is 30-60 minutes. 
     
     
         14 . The method according to  claim 1 , wherein the mass-to-volume ratio of Bayberry leaves to methanol solution is 1:5-20. 
     
     
         15 . The method according to  claim 14 , wherein the mass-to-volume ratio of Bayberry leaves to methanol solution is 1:10. 
     
     
         16 . The method according to  claim 1 , wherein the post-treatment refers to reduced pressure concentration and freeze-drying; the reduced pressure concentration is performed under vacuum rotary evaporation at 37-50° C. 
     
     
         17 . The method according to  claim 1 , wherein the purity of myricetin-3-O-(2″-galloyl)-α-L-rhamnoside monomer and myricetin-3-O-(4″-galloyl)-α-L-rhamnoside monomer is above 98%. 
     
     
         18 . The method according to  claim 1 , wherein the solid phase extraction column is a C18 solid phase extraction column. 
     
     
         19 . The method according to  claim 6 , wherein the volume percentage of the acid is 0.1-3%. 
     
     
         20 . The method according to  claim 8 , wherein 30% alcohol solution is used for the first elution.